SIV strains containing HIV-1 env genes (SHIVenv) have been successfully employed to infect macaques through intravenous and mucosal routes. These macaque models have been crucial for studies on HIV pathogenesis, vaccine, and microbicide testing. However, few SHIVenv strains can maintain stable and prolonged infections. Several challenges are apparent in the testing of anti-HIV-1 microbicides and many of these stem from poor animal models to test efficacy. In the R21 proposal, we have outlined a system to construct and test the infectivity of SHIV based on the env and pol genes of subtype A, B, C, and D from acute/early infections. In aim 1, we will utilize a rapid yeast recombination cloning approach to shuttle approximately 400 HIV-1 env genes into an HIV-1NL4-3 or SIV backbones of mac239 and KB-9. The HIV-1 subtype A, C, and D env genes will be PCR amplified from the endocervix or blood of Ugandan and Zimbabwean women within three months or after three years of infection. Over 20 HIV-1 env chimeric viruses have already been constructed and tested using env genes from these patient samples. HIV and SIV env chimeric viruses will be included in subtype-specific pools if the clone is capable of replication on cell lines expressing human or rhesus CD4/CCR5 (respectively) and in human or rhesus PBMCs (respectively). In aim 2, the pathogenicity of these pools will then be accessed (1) using vaginal explants and (2) through vaginal exposure in macaques. The clones that establish infection in both the explant tissue and macaques can then be reconstituted into the "pathogenic" subtype A, B, C, and D pools for the microbicide studies described in the R33 section of this proposal (aim 3). First, we will determine if higher concentrations of cmpd167 or PSC- RANTES are required to inhibit the "pathogenic" subtype A, B, C, and D pools of HIV or SHIVs (as compared to the standard SHIVSF162-P3) in human or rhesus vaginal explant tissues. We determine the identity of any HIV or SHIV clone(s) that are capable of infection even in the presence of the drug. These specific HIV-1 clones (produced from original DNA clones) can then be tested for sensitivity to CMPD167 and PSC-RANTES and to determine if infection was related to drug resistance. Finally and most importantly, microbicides CMPD167 and PSC-RANTES will be vaginally applied to rhesus macaques prior to exposure with the "infectious" subtype A, B, C, and D pools as well as the standard SHIVSF162-P3. We suspect that the majority of the treated macaques will be protected from SHIVSF162-P3 infection. In contrast, the protective effects of the microbicides may be reduced and that in some animals, a slight delay in viremia (as compared to untreated animals) may be the result of infection by specific clone in the SHIV pool with reduced sensitivity to CMPD167 and PSC-RANTES. Vaginal microbicides provide an excellent method to protect women from HIV-1 infection but testing these products prior to human use remains a challenge. A monkey species (e.g. Rhesus macaques) and virus cousin of HIV-1 (SHIV) are used to test the level of protection by these compounds. In this proposal, we have designed new SHIVs that are more closely related to HIV-1 and provide more stringent testing of microbicides for future human use.